Hi! This is an attempt to write simply about things I feel passionate about. My name is Judith Recht and I am a scientist by training, a later-in-life mother, and an expat in Bangkok, Thailand and Recife, Brazil (~4 years in each country) now back in the US. I was born in one country (USA) grew up in another (Venezuela) raised by Argentine parents and moved around four more times (NYC to Bangkok to Recife to Maryland). This website and blog is for those of you who might be interested in the diverse topics mentioned below (blog entries starting January 2013) as well as the ones here in my homepage:
1) animal models for human disease (mouse model)
2) drug-resistant infections
3) how doing molecular biology research has dramatically changed in recent years
4) the awesome PCR
5) publishing research from less developed countries
6) telomeres, aging and cancer
7) why you should know about biofilms
8) are we (especially women) more chimeric and mosaical than we thought?
9) do you know what your blood type is?
10) praising women scientists
11) malaria
12) genetic testing for disease risk
13) chikungunya: an emerging tropical disease in the Americas
14) Chagas: a neglected tropical disease in the Americas
15) those amazing cell powerhouses called mitochondria
16) tuberculosis
17) we all carry around a HUGE microbiome (what the heck is that?)
18) Medicine approaches focused on boosting our defenses: Immunotherapy as a promising treatment for cancer
19) A new viral threat to Latin America: Zika virus
20) endocrine disruptors are everywhere- why you should be concerned
21) levels of complexity within us
22) antibiotics: the good and the bad
23) perimenopause: the 0-10 years period before menopause
24) thanking yeast
25) the real potential of the new genome editing technology CRISPR-CAS9
26) Jumping pieces in our DNA called transposons
27) pharmacogenetics and personalized medicine: can our genes predict how we will respond to medication?
28) appreciate your ATP
29) writing a scientific manuscript for peer review (with tips for non-English native speakers)
30) basic principles of immunology are used in medical tests that allow you to know your disease, pregnancy or blood type status
31) why your circadian rhythms are important for your health
32) get to know your vitamins
33) vaccines: why they help everybody (not just the vaccinated) but only when most people get them
34) what are clinical trials and why do they take so long?
35) thanking Henrietta: what makes cancer cells malignant is good for research
36) Vaccine refusal is harming disease eradication
37) electric bacteria or the electromicrobiology field
38) senolytics or the search for a longer healthier life
39) Do you hear ringing clicking buzzing in your ears? A common and interesting symptom called tinnitus
40) Understanding domestication
41) Zoonotic diseases (transmitted to humans from other animals) are more common than you might think
42) Menopause: the HRT dilemma
43) The single cell analysis breakthrough
44) Thanking llamas and other camelids for their nanobodies a potential treatment for covid 19 and other diseases
45) COVID-19 vaccines January 2021
46) Citizen Science: get involved
47) Ocean Acidification: why should we care
1) animal models for human disease (mouse model)
2) drug-resistant infections
3) how doing molecular biology research has dramatically changed in recent years
4) the awesome PCR
5) publishing research from less developed countries
6) telomeres, aging and cancer
7) why you should know about biofilms
8) are we (especially women) more chimeric and mosaical than we thought?
9) do you know what your blood type is?
10) praising women scientists
11) malaria
12) genetic testing for disease risk
13) chikungunya: an emerging tropical disease in the Americas
14) Chagas: a neglected tropical disease in the Americas
15) those amazing cell powerhouses called mitochondria
16) tuberculosis
17) we all carry around a HUGE microbiome (what the heck is that?)
18) Medicine approaches focused on boosting our defenses: Immunotherapy as a promising treatment for cancer
19) A new viral threat to Latin America: Zika virus
20) endocrine disruptors are everywhere- why you should be concerned
21) levels of complexity within us
22) antibiotics: the good and the bad
23) perimenopause: the 0-10 years period before menopause
24) thanking yeast
25) the real potential of the new genome editing technology CRISPR-CAS9
26) Jumping pieces in our DNA called transposons
27) pharmacogenetics and personalized medicine: can our genes predict how we will respond to medication?
28) appreciate your ATP
29) writing a scientific manuscript for peer review (with tips for non-English native speakers)
30) basic principles of immunology are used in medical tests that allow you to know your disease, pregnancy or blood type status
31) why your circadian rhythms are important for your health
32) get to know your vitamins
33) vaccines: why they help everybody (not just the vaccinated) but only when most people get them
34) what are clinical trials and why do they take so long?
35) thanking Henrietta: what makes cancer cells malignant is good for research
36) Vaccine refusal is harming disease eradication
37) electric bacteria or the electromicrobiology field
38) senolytics or the search for a longer healthier life
39) Do you hear ringing clicking buzzing in your ears? A common and interesting symptom called tinnitus
40) Understanding domestication
41) Zoonotic diseases (transmitted to humans from other animals) are more common than you might think
42) Menopause: the HRT dilemma
43) The single cell analysis breakthrough
44) Thanking llamas and other camelids for their nanobodies a potential treatment for covid 19 and other diseases
45) COVID-19 vaccines January 2021
46) Citizen Science: get involved
47) Ocean Acidification: why should we care
We have now, available on Amazon, both an English and a Spanish version of a grief journal: Write Through It and Procésalo Escribiendo respectively - check them out!
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Rambling thoughts on genetics, histones and epigenetics from someone who fell for them more than 20 years ago
Soon (meaning one of these years!) I trust the Nobel prize in physiology or medicine will be awarded to the field of epigenetics- to the pioneers who are still alive. That will be an exciting recognition to this once neglected field. Epigenetic phenomena such as histone and DNA modifications (acetylation, methylation) are not so unheard of nowadays due to their roles in several diseases including cancer. New treatments now focus on these epigenetic targets. But I am getting ahead of myself and perhaps scaring you with weird terminology, because this story starts much earlier when I was in college in Caracas, Venezuela….
In the early 1990s I was a University graduate with a college degree in “cellular biology” from Venezuela trying desperately to get accepted into a PhD program in the US, where I happen to be born so I had the precious citizenship required to get a fellowship. During my undergrad in biology, I started with animal and plant biology courses, ecology and others which were interesting and exiting, including laboratory sessions that lasted several hours and involved dissecting samples and looking at them under the microscope and field trips to beautiful regions in Venezuela such as different types of beaches (sandy, coral and rocky beaches) as well as rain forest, lakes and others. Most of my friends ended up focusing on ecology, marine biology or tropical parasitology. Once I started taking genetics, microbiology and biochemistry courses, I realized I was a more molecularly-oriented kind of biologist.
While waiting for an interview to start a PhD program in the US, I worked in a private clinical center in Caracas as part of a Chagas disease research group in the same laboratory where other groups were doing pioneering tests and procedures at the time, including amniocentesis to provide “karyotyping” results to pregnant mothers, and IVF (in vitro fertilization) for infertile couples when these were both fields in their very exciting beginnings.
I take a detour here to tell you how exciting to watch this was and to explain as simply as I can, how they were done. For an amniocentesis, after the amniotic liquid was extracted from the pregnant mother by the doctor with a syringe, the sample was processed by a technician specialized in “culturing” these cells (growing them in an appropriate medium so the cells can divide and multiply to result in a good-enough cellular mass that can be then analyzed) for a few weeks to then do chromosomes staining and counting. Chromosomes are discrete units inside the nucleus of each cell which contain DNA, and within each chromosome there are many different genes. The work done by the technicians in our lab in the early ‘90s looking at these chromosomes under the microscope, taking pictures and then cutting from them the chromosome pairs individually to put on a piece of paper for the results report (karyotype, see Figure below) was, to me, the work of an artist who knew and loved its craft. The staff recognized the chromosomes without any help from the book or manual. Some had to write across the cover of the results folder “does not want to know sex” because the fetus’ gender could be easily spotted by the doctor by just looking at the pasted chromosomes: 23 pairs in total, the last one (pair 23) is the sexual one which consists of either 2 similar ones (XX, a female) or two different ones (a bigger X and a smaller Y, a male). Nowadays, all you hear from your obstetrician is “everything is OK” or not…
IVF is a very demanding process that couples go through (mostly tough on the females) when they can not conceive naturally over an extended period of time. The woman is induced to “super ovulate” with the help of powerful female hormones, the eggs produced are extracted by the doctor, and the male is asked to produce a sample of sperm, which might be “processed” in the lab a bit to then be incubated with the eggs to result in fertilization. The fertilization process by which one sperm (male part) merges with an egg (female part) is followed under the microscope to monitor how many embryos are formed and whether they are “good” or not; this includes a few cell divisions of the now formed embryo. One of the trickiest things with IVF from the beginning was an important decision the doctors had to make: how many of these embryos to inject in the woman for “implantation” in the uterus. The success rate was not very high back then, so a few (sometimes 4-5) were used, which led to multiple pregnancies like twins, triplets etc. There was some talk already in the early ‘90s regarding the possibility of testing individual cells from IVF embryos for abnormalities before trying to use the embryos for implantation into the female uterus, which back then sounded a little like sci-fi. The remaining embryos (if any) as well as the retrieved eggs which were not used, were frozen at -80°C to keep them available in case the cycle was not successful and the couple wanted to try again, in which case the frozen ones could be thawed and used to attempt again fertilization/implantation.
I had decided during my undergraduate thesis project (which in some Latin American countries is the equivalent of a masters in the US including 5-6 years of both courses and a laboratory research thesis project guided by a mentor) that I was interested in what was then known as “genetic regulation”. Cell and molecular studies indicated that a lot of what happens inside our cells and tissues was under the control of things that in the end were responsible for turning genes ON or OFF. This is known in genetics as gene “induction” or “repression” respectively. At the time all of this was thought to be “regulated” by special proteins called “transcription factors”. Transcription is the cellular process by which the information in our genes (DNA, which is double-stranded with one of these strands acting as a “template”) is “transcribed” into a different molecule (messenger RNA, single-stranded) which then has to travel from the nucleus of the cell into the cytoplasm to be “translated” into a protein, which is a whole different story…
Genetics and mutations- they are so much fun!
Since I was a little girl I have greatly enjoyed detective stories. I devoured Agatha Christie’s crime novels and short stories- I liked them all, no matter whether the crimes were solved by Mr Poirot or Miss Marple, her two great detective characters. I think the process by which scientists attempt to solve the problems they analyze is similar to being a detective, the difference consisting mainly on how the clues are generated. The detective has to find clues left at the crime scene, as well as at other places and people related to the crime, whereas scientists generate the clues that come from the experiments they perform, which are designed to answer the questions and hypotheses raised. In the laboratory, new results lead to new questions that then need new clues to be answered. These are all solved by logical reasoning based on the results obtained, and yes, sometimes intuition… although I think intuition comes in part from experience acquired by solving prior mysteries.
When one is looking at and trying to figure out how things work inside cells, more “molecularly” speaking, there are two (once opposing, now complementary) approaches, which depending on how much is known and how much one wants to find out, can be very useful. One is biochemistry and the other one is genetics.
Genetics allows you, among other things, to ask what happens in the ABSENCE of something you’re interested in to then infer what the PRESENCE of it actually does. For example, take gene X is a mystery to you. You want to find out what function it performs, i.e. which protein it encodes and what this protein does. By specific methods that continue to develop and improve year after year, one can generate specific “mutations” in a target gene in different biological organisms. One of these gene manipulations, the simplest one in terms of output generated, is to “delete” the gene. By looking at what happens to the cells when this gene is eliminated, one can infer what the function of the gene is when present. Unless…. if the gene encodes an “essential” protein, meaning one that is essential for the life of the cell, then a deletion will result in a dead cell in which case you also get valuable information. If this is the case, there are ways to generate a mutation that alters the gene but does not eliminate it, leaving a little function left, and again based on what you obtain you can deduce the function of the gene. For example, if once you generate a mutation the previously motile cell can no longer move, the function of the gene has to do with cell motility. If the cell shape is altered, then the gene has to do with cellular shape, and so on…
I had decided during my undergraduate thesis project (which in some Latin American countries is the equivalent of a masters in the US including 5-6 years of both courses and a laboratory research thesis project guided by a mentor) that I was interested in what was then known as “genetic regulation”. Cell and molecular studies indicated that a lot of what happens inside our cells and tissues was under the control of things that in the end were responsible for turning genes ON or OFF. This is known in genetics as gene “induction” or “repression” respectively. At the time all of this was thought to be “regulated” by special proteins called “transcription factors”. Transcription is the cellular process by which the information in our genes (DNA, which is double-stranded with one of these strands acting as a “template”) is “transcribed” into a different molecule (messenger RNA, single-stranded) which then has to travel from the nucleus of the cell into the cytoplasm to be “translated” into a protein, which is a whole different story…
Genetics and mutations- they are so much fun!
Since I was a little girl I have greatly enjoyed detective stories. I devoured Agatha Christie’s crime novels and short stories- I liked them all, no matter whether the crimes were solved by Mr Poirot or Miss Marple, her two great detective characters. I think the process by which scientists attempt to solve the problems they analyze is similar to being a detective, the difference consisting mainly on how the clues are generated. The detective has to find clues left at the crime scene, as well as at other places and people related to the crime, whereas scientists generate the clues that come from the experiments they perform, which are designed to answer the questions and hypotheses raised. In the laboratory, new results lead to new questions that then need new clues to be answered. These are all solved by logical reasoning based on the results obtained, and yes, sometimes intuition… although I think intuition comes in part from experience acquired by solving prior mysteries.
When one is looking at and trying to figure out how things work inside cells, more “molecularly” speaking, there are two (once opposing, now complementary) approaches, which depending on how much is known and how much one wants to find out, can be very useful. One is biochemistry and the other one is genetics.
Genetics allows you, among other things, to ask what happens in the ABSENCE of something you’re interested in to then infer what the PRESENCE of it actually does. For example, take gene X is a mystery to you. You want to find out what function it performs, i.e. which protein it encodes and what this protein does. By specific methods that continue to develop and improve year after year, one can generate specific “mutations” in a target gene in different biological organisms. One of these gene manipulations, the simplest one in terms of output generated, is to “delete” the gene. By looking at what happens to the cells when this gene is eliminated, one can infer what the function of the gene is when present. Unless…. if the gene encodes an “essential” protein, meaning one that is essential for the life of the cell, then a deletion will result in a dead cell in which case you also get valuable information. If this is the case, there are ways to generate a mutation that alters the gene but does not eliminate it, leaving a little function left, and again based on what you obtain you can deduce the function of the gene. For example, if once you generate a mutation the previously motile cell can no longer move, the function of the gene has to do with cell motility. If the cell shape is altered, then the gene has to do with cellular shape, and so on…
On the other hand, biochemistry allows you to work with the PROTEIN (the product of the gene) and ask directly what function it performs. Biochemistry is also a more “in vitro” assay (in the test tube). This means you can take a “purified” protein (which may come from live cells that were lysed to release their contents including the proteins of interest) and put it in a test tube in an appropriate buffer and conditions and test whether it interacts with or modifies other proteins. You can look at molecular sizes of the proteins and domains, you can test interactions between different proteins. You can test models by adding or eliminating things in the test tube that should help or affect the phenomenon you are looking at. Genetics is a more “in vivo” approach (inside the cell) as you are still looking at what happens with the LIVE cell- although it has been manipulated genetically; it is not in the test tube.
There are two strategies to use genetically to make mutations that can give us a lot of information about a cellular process or gene. The first strategy applies when we know the gene we are interested in. This means we might know the location in the chromosome, and more importantly the gene’s DNA sequence. With the appropriate tools we can “target” this gene of interest and make specific mutations in it. We can either delete it (remove the gene completely), or modify it to make less (or more!) amount of the protein it encodes (let’s not forget that genes, when “expressed” result in proteins being made which perform different functions at specific locations). The deletion will not work when the gene is essential for cell survival or growth. The results of each of these manipulations, in terms of what happens in the cell, are very useful in the process of elucidating the gene’s function.
What we look at once the gene is mutated is called the “phenotype”. This phenotype could be the cell shape, color, motility, or any other behavior this gene’s protein product is involved in. The mutation itself is the “genotype”, which is responsible for the phenotype we see. By analyzing the results from different mutations in the same gene we can sometimes deduce the function of the gene.
There are two strategies to use genetically to make mutations that can give us a lot of information about a cellular process or gene. The first strategy applies when we know the gene we are interested in. This means we might know the location in the chromosome, and more importantly the gene’s DNA sequence. With the appropriate tools we can “target” this gene of interest and make specific mutations in it. We can either delete it (remove the gene completely), or modify it to make less (or more!) amount of the protein it encodes (let’s not forget that genes, when “expressed” result in proteins being made which perform different functions at specific locations). The deletion will not work when the gene is essential for cell survival or growth. The results of each of these manipulations, in terms of what happens in the cell, are very useful in the process of elucidating the gene’s function.
What we look at once the gene is mutated is called the “phenotype”. This phenotype could be the cell shape, color, motility, or any other behavior this gene’s protein product is involved in. The mutation itself is the “genotype”, which is responsible for the phenotype we see. By analyzing the results from different mutations in the same gene we can sometimes deduce the function of the gene.
We could also grow the cells in different conditions (different nutrients, humidity conditions, or temperatures for example) and look at the different phenotypes for the same mutation in these conditions. Actually, many “accidental” discoveries in science have come about by trying something that was originally designed for one experiment or disease, in another disease or condition, or by something that went wrong in the experiment leading to a totally unexpected discovery. Two notable examples are the discoveries of the properties of the widely used antibiotic penicillin and the more recently licensed Viagra. Alexander Fleming, a Scottish Nobel prize recipient serendipitously discovered penicillin in 1928 in his laboratory at St. Mary's Hospital in London when he noticed a blue-green mold contaminant growing on a Petri dish containing Staphylococcus that he had left open by mistake. And here is where the amazing scientist-detective mentality was revealed: most scientists would discard the plate as a contamination, but Fleming noticed a halo of inhibition around the contaminant growth, a clear area where nothing else grew. This made him hypothesize that the mold released a substance that repressed bacterial growth around it, and research continued on this subject to result in the purification and wide use of penicillin. Viagra was a candidate drug originally developed by Pfizer to treat angina which resulted in the a widely prescribed drug for erectile dysfunction after an unexpected side effect (erections lasting long time) was reported by volunteers involved in testing the drug in earlier trials.
However, let’s say we are interested in a cellular process but don’t know the genes involved and would like to find out more. This is where the second strategy to make and look at different mutations comes in. For this purpose, we can perform what’s called a “genetic screen”. There are different ways to generate mutants, and in this case one would go after a mutant “library” which is a collection of mutations in different individual genes, ideally one each in all the genes in the cell which can be mutated without resulting in death or very sick cells. The screen conducted is for a phenotype we are interested in- let’s say cell motility. We would take each member of the mutant library and “grow” them on an agar plate where we can see movement. We are interested in detecting those that can NOT move. These are then the cells that are mutant in genes that normally would ALLOW the cells to move. Then we can make use of modern high-tech procedures and sequence the genes mutated in these cells. We can "see" which proteins they encode, and what these proteins do (if it is known- and if not then we can "purify" them and test them “in vitro” using biochemistry).
However, let’s say we are interested in a cellular process but don’t know the genes involved and would like to find out more. This is where the second strategy to make and look at different mutations comes in. For this purpose, we can perform what’s called a “genetic screen”. There are different ways to generate mutants, and in this case one would go after a mutant “library” which is a collection of mutations in different individual genes, ideally one each in all the genes in the cell which can be mutated without resulting in death or very sick cells. The screen conducted is for a phenotype we are interested in- let’s say cell motility. We would take each member of the mutant library and “grow” them on an agar plate where we can see movement. We are interested in detecting those that can NOT move. These are then the cells that are mutant in genes that normally would ALLOW the cells to move. Then we can make use of modern high-tech procedures and sequence the genes mutated in these cells. We can "see" which proteins they encode, and what these proteins do (if it is known- and if not then we can "purify" them and test them “in vitro” using biochemistry).
As you might know, there are many diseases including different types of cancer, where the disease has been shown to correlate with specific mutations. In patients, molecular assays such as PCR can be used to “read” the DNA sequence at the place where the mutation that imposes the risk for the disease is located. In this case, the mapped mutation (genotype) confers the sick phenotype. If this mutation is known to occur with a certain frequency in a family (for example in related women resulting in higher risk of breast and ovarian cancer the mutations are in known genes called BRCA1 and BRCA2) it increases the risk for younger members who can get tested and if the mutation is confirmed, they can opt to take drastic preventive measures such as mastectomy for breast cancer. In humans there are many factors affecting this risk (exercise, smoking, drinking, exposure to toxins from the environment, stress, other diseases and many etcs!) so evaluating them can be very complicated. In the laboratory and with model organisms, we can control many of the variables to assess specifically the effect of the mutation of interest. For human diseases, there are many models in mice including for Alzehimer’s, diabetes, cancer, and obesity. Mice live shorter lives and one can engineer mutations in them which can not be made in humans, for example the equivalent mutation which in human patients results in the disease, and study the animals in detail to get more information regarding the disease, and hopefully afterwards test candidate treatments and vaccines in the mouse model of the disease. Most treatments that make it to clinical trials have been previously tested at the pre-clinical level in animals. We can also infect animals with virus or bacteria and then test possible drugs, or inoculate them with candidate vaccines and then test the protective effect against the infection (check out my first blog entry on animal models for human disease).
PCR (polymerase chain reaction) is now used in other fields such as forensics and criminology to identify dead people or criminal suspects- by using something as small as a piece of hair left at the crime scene or semen sample from a rape, PCR-based assays will result in a "DNA fingerprint" that is unique to the person it comes from. Thanks to popular TV shows that now mention these and other molecular assays, people are now more familiar with the terminology. In addition, PCR can unequivocally offer answers when used for paternity tests in which the mother, putative father and the child provide samples for analysis; the most common sample for these tests is saliva, taken by oral swab.
Another great tool to help us figure out what happens inside the cells is "cell biology" which includes using microscopic techniques to visualize live cells or dead cells that have been "fixed" to preserve their shape and contents. There are amazing procedures now in the molecular biology field that allow researchers to "tag" a specific protein with a flourescent dye which makes the tagged protein look either green, red or cyan when exposed to certain light wavelengths under the "fluorescence microscope". A very popular tag is GFP or "green fluorescent protein" (a protein naturally found in jellyfish and which was the subject of a Nobel prize in Chemistry awarded to its discoverer and developers in 2008). Using this approach, we can tag a protein and see where inside the cell it localizes, also look at different times of the cell cycle to see if the protein travels to different locations, or we can put the tagged protein inside a mutant cell in a gene that we suspect has something to do with the tagged protein, for example its subcellular localization, and see whether this is affected in the mutant. There are also different color tags that can be used now, so one can tag more than one protein and look at interactions between them inside a live cell.
PCR (polymerase chain reaction) is now used in other fields such as forensics and criminology to identify dead people or criminal suspects- by using something as small as a piece of hair left at the crime scene or semen sample from a rape, PCR-based assays will result in a "DNA fingerprint" that is unique to the person it comes from. Thanks to popular TV shows that now mention these and other molecular assays, people are now more familiar with the terminology. In addition, PCR can unequivocally offer answers when used for paternity tests in which the mother, putative father and the child provide samples for analysis; the most common sample for these tests is saliva, taken by oral swab.
Another great tool to help us figure out what happens inside the cells is "cell biology" which includes using microscopic techniques to visualize live cells or dead cells that have been "fixed" to preserve their shape and contents. There are amazing procedures now in the molecular biology field that allow researchers to "tag" a specific protein with a flourescent dye which makes the tagged protein look either green, red or cyan when exposed to certain light wavelengths under the "fluorescence microscope". A very popular tag is GFP or "green fluorescent protein" (a protein naturally found in jellyfish and which was the subject of a Nobel prize in Chemistry awarded to its discoverer and developers in 2008). Using this approach, we can tag a protein and see where inside the cell it localizes, also look at different times of the cell cycle to see if the protein travels to different locations, or we can put the tagged protein inside a mutant cell in a gene that we suspect has something to do with the tagged protein, for example its subcellular localization, and see whether this is affected in the mutant. There are also different color tags that can be used now, so one can tag more than one protein and look at interactions between them inside a live cell.
GFP inclusions in McCoy cells infected with C. trachomatis. Panel A shows untransformed C. trachomatis under white light (arrows indicate inclusions). Panel B is the same image under blue light. Panel C shows C. trachomatis transformed with pGFP::SW2 under white light, and panel D is the same field under blue light.
Modified Figure 6 from: Wang Y, Kahane S, Cutcliffe LT, Skilton RJ, et al. (2011) Development of a Transformation System for Chlamydia trachomatis: Restoration of Glycogen Biosynthesis by Acquisition of a Plasmid Shuttle Vector. PLoS Pathog 7(9): e1002258. doi:10.1371/journal.ppat.1002258
http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002258
Then came histones!
There was in the early ‘90s a relatively small but growing group of investigators that started focusing on proteins that DNA was bound to called “histones”. Histones were known to be present in a complex called “chromatin” which referred to the structural unit formed by the DNA and histones together, but they were viewed as having a mere structural role, keeping the DNA heavily compacted inside the eukaryotic nucleus. If the DNA inside one human cell could be stretched out, it would span about 6 feet (~1.8 meters). In contrast, bacteria, which belong to the group of organisms known as prokaryotes, do not have nuclei: the word karyon comes from Greek meaning nucleus, and “pro” means before.
The histone people argued that histones had a much more active role in genetic regulation, in activating and repressing genes, and this became more apparent when new studies revealed that these histones were “post-translationally modified”. These modifications, which can be viewed as “flavors” or variations of the basic histone protein forms, and some of which occur in a very dynamic fashion meaning they can be put on and removed by specific protein modifiers, make them exquisitely diverse. I’ll describe for now only the ones known as “core” histones - there are several other ones.
An interesting fact about histones is that they are the proteins that are most evolutionarily conserved throughout eukaryote organisms, from plants to humans, especially the core histones H3 and H4. When a protein is conserved (meaning its amino acid composition which determines its 3D structure has not diverged from one organism/species to another much) it is thought to have a crucial and very important biological role. Protein conservation is actually revealed (and kept) in the gene's DNA sequence. Evolution allows the genes encoding the proteins to vary via the appearance of mutations which can be “selected” when the new proteins perform better, a process that takes many generations. This is particularly important when environmental conditions change and perhaps the old protein forms are no longer the best performers for their biological function under the new conditions. The number of histone gene copies in eukaryotes is also much higher than for genes encoding other proteins; in some instances there might be over 50 gene copies coding for core histones within once cell, and they may be present in gene clusters on different chromosomes in mammals. Because histone proteins are distributed all over the DNA which wraps around them, their intracellular numbers are very high compared to other cellular proteins. We can think of them as having a structural role in helping compact DNA and additionally performing critical roles for several cellular processes such as gene expression, DNA replication, and repair from DNA damage.
Chromatin is the term we use to refer to the DNA and the proteins that bind to it such as histones, to result in compaction of this DNA inside the cell nucleus. The basic unit of chromatin is known as the “nucleosome”, composed of about 146 base pairs of DNA (consisting of 4 possible base types: G, C, T or A) wrapped almost twice around a histone octamer (see Figure below). This octamer is formed by 4 histone dimers. The inner core is a tetramer formed by two heterodimers of histones called H3 and H4, onto which two dimmers of histones H2A and H2B are deposited during the DNA replication process that happens once per cell cycle and possibly in a much more dynamic fashion at other times of the cell cycle when induction or repression of transcription occur. Additional “linker” histone proteins such as histone H1 bind to nucleosomes and serve as linkers between them to result in further compaction of the histone fiber. Not all eukaryotes have linker histones. Most of the first histone modifications to be studied and characterized occur in the “tails” of the core histones. These tails are flexible portions of the histones located mostly at the N-termini of the core histones and which protrude a bit, coming out of the tightly linked histone-DNA domains and are therefore more accessible to the proteins that can modify them by adding acetyl, methyl and other groups to specific amino acid residues.
There was in the early ‘90s a relatively small but growing group of investigators that started focusing on proteins that DNA was bound to called “histones”. Histones were known to be present in a complex called “chromatin” which referred to the structural unit formed by the DNA and histones together, but they were viewed as having a mere structural role, keeping the DNA heavily compacted inside the eukaryotic nucleus. If the DNA inside one human cell could be stretched out, it would span about 6 feet (~1.8 meters). In contrast, bacteria, which belong to the group of organisms known as prokaryotes, do not have nuclei: the word karyon comes from Greek meaning nucleus, and “pro” means before.
The histone people argued that histones had a much more active role in genetic regulation, in activating and repressing genes, and this became more apparent when new studies revealed that these histones were “post-translationally modified”. These modifications, which can be viewed as “flavors” or variations of the basic histone protein forms, and some of which occur in a very dynamic fashion meaning they can be put on and removed by specific protein modifiers, make them exquisitely diverse. I’ll describe for now only the ones known as “core” histones - there are several other ones.
An interesting fact about histones is that they are the proteins that are most evolutionarily conserved throughout eukaryote organisms, from plants to humans, especially the core histones H3 and H4. When a protein is conserved (meaning its amino acid composition which determines its 3D structure has not diverged from one organism/species to another much) it is thought to have a crucial and very important biological role. Protein conservation is actually revealed (and kept) in the gene's DNA sequence. Evolution allows the genes encoding the proteins to vary via the appearance of mutations which can be “selected” when the new proteins perform better, a process that takes many generations. This is particularly important when environmental conditions change and perhaps the old protein forms are no longer the best performers for their biological function under the new conditions. The number of histone gene copies in eukaryotes is also much higher than for genes encoding other proteins; in some instances there might be over 50 gene copies coding for core histones within once cell, and they may be present in gene clusters on different chromosomes in mammals. Because histone proteins are distributed all over the DNA which wraps around them, their intracellular numbers are very high compared to other cellular proteins. We can think of them as having a structural role in helping compact DNA and additionally performing critical roles for several cellular processes such as gene expression, DNA replication, and repair from DNA damage.
Chromatin is the term we use to refer to the DNA and the proteins that bind to it such as histones, to result in compaction of this DNA inside the cell nucleus. The basic unit of chromatin is known as the “nucleosome”, composed of about 146 base pairs of DNA (consisting of 4 possible base types: G, C, T or A) wrapped almost twice around a histone octamer (see Figure below). This octamer is formed by 4 histone dimers. The inner core is a tetramer formed by two heterodimers of histones called H3 and H4, onto which two dimmers of histones H2A and H2B are deposited during the DNA replication process that happens once per cell cycle and possibly in a much more dynamic fashion at other times of the cell cycle when induction or repression of transcription occur. Additional “linker” histone proteins such as histone H1 bind to nucleosomes and serve as linkers between them to result in further compaction of the histone fiber. Not all eukaryotes have linker histones. Most of the first histone modifications to be studied and characterized occur in the “tails” of the core histones. These tails are flexible portions of the histones located mostly at the N-termini of the core histones and which protrude a bit, coming out of the tightly linked histone-DNA domains and are therefore more accessible to the proteins that can modify them by adding acetyl, methyl and other groups to specific amino acid residues.
Nucleosome structure. The 4 core histones H3, H4, H2A and H2B are schematically shown in different colors to depict one nucleosome with DNA wrapped around twice. The DNA exiting the nucleosome on the sides is called linker DNA which might be bound by linker histones like H1 (not shown here) and contact neighbor nucleosomes. The flexible histone tails are also shown leaving the core histone-DNA complex.
Nucleosomes, when further compacted via tight interactions with other nucleosomes, linker DNA and linker histones, form chromatin fibers (see first Figure below). The highest level of compaction is needed for chromosomes during metaphase, showing the karyotype we mentioned above. This is the only moment in the life of the cell (cell cycle) when chromosomes (and therefore the karyotype) are visible under the microscope. During other times of the cell cycle, chromatin is in a more diffuse and “open” state, and condensed chromosomes are not seen. The second figure below shows the appearance of chromatin in onion cells in the different phases of the cell cycle, with maximum compaction in discrete "chromosome" forms appreciated at metaphase.
From DNA to chromatin compaction. Compaction is mediated by histone proteins (from Abcam: http://www.abcam.com/index.html?pageconfig=resource&rid=9). The nucleosomes with linker DNA but no linker histones bound is shown in the middle structure known as “beads on a string” fiber of 10 nm thickness; addition of linker histones leads to a more compacted chromatin fiber of ~ 30 nm (the bottom structure).
Until the roles of chromatin and histones in gene expression were more widely recognized, the experiments done with DNA “in vitro” did not include “chromatin” as such, they were done with what’s called “naked DNA” (not including histones in the test tube). Later on, more “natural” DNA substrates were used including DNA wrapped around histones forming chromatin, and then different positive (induction) or negative (repression) effects on transcription were seen in assays that were not detected before. The main effect was that chromatin was usually inhibitory to transcription as opposed to naked DNA. The number of individual histone modifications on different amino acid residues and the number of combinations of these that can be found in one nucleosome result in what is known as a “histone code” which can exquisitely modulate gene expression. Some modifications are specifically associated with gene activation, some with repression. Sometimes a long stretch of nucleosomes needs to be modified in a certain way to result in silencing of the genes contained in this region.
You could be asking yourself at this point, what happens if a gene that needs to be expressed (i.e. made into RNA so it can be made into a protein such as a hormone, enzyme, structural constituent of a tissue etc etc) is buried deep into chromatin? How do transcription factors get access to it? This is where sometimes histone modifications play a critical role. The first histone modification studied in detail was acetylation, which consists on the addition of acetyl groups to lysine residues; lysine is one of the amino acids that form proteins and is positively charged. The DNA backbone is negatively charged due to the phosphates. Even back in the 1960s, a pioneer and visionary Rockefeller University researcher, Vincent Allfrey, showed and published beautiful biochemistry work demonstrating the existence of acetylated lysine residues in the amino termini of the H3 and H4 core histones. The authors of this publication hypothesized back then that these chemically modified (acetylated) histones could be neutralizing the positive charge of lysines and therefore weakening their interaction with the negatively charged DNA, leaving chromatin more accessible to gene activation and expression.
Now we know there are specific enzymes responsible for adding or removing acetyl groups from histone lysines: HATs (histone-acetyltransferases) and HDACs (histone-deacetylases) which modulate the acetylation state of chromatin and could affect compaction by their role on loosening the histone-DNA interaction (see Figure below).
You could be asking yourself at this point, what happens if a gene that needs to be expressed (i.e. made into RNA so it can be made into a protein such as a hormone, enzyme, structural constituent of a tissue etc etc) is buried deep into chromatin? How do transcription factors get access to it? This is where sometimes histone modifications play a critical role. The first histone modification studied in detail was acetylation, which consists on the addition of acetyl groups to lysine residues; lysine is one of the amino acids that form proteins and is positively charged. The DNA backbone is negatively charged due to the phosphates. Even back in the 1960s, a pioneer and visionary Rockefeller University researcher, Vincent Allfrey, showed and published beautiful biochemistry work demonstrating the existence of acetylated lysine residues in the amino termini of the H3 and H4 core histones. The authors of this publication hypothesized back then that these chemically modified (acetylated) histones could be neutralizing the positive charge of lysines and therefore weakening their interaction with the negatively charged DNA, leaving chromatin more accessible to gene activation and expression.
Now we know there are specific enzymes responsible for adding or removing acetyl groups from histone lysines: HATs (histone-acetyltransferases) and HDACs (histone-deacetylases) which modulate the acetylation state of chromatin and could affect compaction by their role on loosening the histone-DNA interaction (see Figure below).
Nucleosomes consisting each of DNA (blue line) wrapped around an octamer of histone proteins (lilac circle) with protruding histone tails (purple lines) with acetyl groups (red dots) added by the HAT enzymes are more “open” or spaced apart than the same nucleosomes which have undergone removal of the acetyl groups by the HDAC enzymes.
Once I learned that all or most cells and tissues in our bodies contain EXACTLY the same DNA sequence (the same genes, in the same chromosomes) I was even more amazed at the exquisitely diverse intracellular regulation that must occur at the level of genetic control to allow activation of specific genes that need to be activated in one cell and repression of the same gene in another (NOTE: see blog entry september 2013 on chimera/mosaic humans (especially women) showing that there are actually groups of cells/tissues that do not contain the same DNA sequence). If you think of a liver cell versus heart, eye, kidney, lungs.... Many signals that make tissue-specific cells produce enzymes and hormones (insuline by the pancreas, thyroid hormone by the thyroid gland, sex homones by the ovaries and testes) come from the outside of the cell and go inside, but eventually the effect is to turn on or off genes that result in the making of specific enzymes or hormones that our body needs made.
Because cells in our bodies are hard to manipulate genetically (for example for histones, there are many gene copies for the same histone protein in each cell), scientists use “model organisms”. Historically, a simple model organism to do genetics has been the budding yeast Saccharomyces cerevisiae. Among other advantages, it is unicellular. It has fewer copies of important genes, including histones (two for each of the core histones) and there are relatively easy methods to generate different types of mutations in them. It is easy to grow in the lab, whether in liquid media or on agar plates, and most importantly, it has two “mating” types, “a” and “alpha". These two “flavors” can grow separately as haploid organisms (X number of chromosomes), or mate with each other to result in diploids (2X number of chromosomes). These sexual states that can mate allow the researcher to “cross” different mutations of interest to look at interactions by mating an “a” cell containing mutation Y with an “alpha” cell containing mutation Z (provided none of these Y and Z mutations results in dead or very sick cells). When looking at two (or more!) different mutations together the results are more diverse and can provide more information of the relation between two different proteins and their roles in a particular cellular process. It was this yeast species (which is also a widely used "brewing yeast" to make cider and beer!) that was first used to elucidate the role of specific histone modifications, as one could make individual mutations and also combo mutants to study interactions between different modifications in the same or different histones.
Because cells in our bodies are hard to manipulate genetically (for example for histones, there are many gene copies for the same histone protein in each cell), scientists use “model organisms”. Historically, a simple model organism to do genetics has been the budding yeast Saccharomyces cerevisiae. Among other advantages, it is unicellular. It has fewer copies of important genes, including histones (two for each of the core histones) and there are relatively easy methods to generate different types of mutations in them. It is easy to grow in the lab, whether in liquid media or on agar plates, and most importantly, it has two “mating” types, “a” and “alpha". These two “flavors” can grow separately as haploid organisms (X number of chromosomes), or mate with each other to result in diploids (2X number of chromosomes). These sexual states that can mate allow the researcher to “cross” different mutations of interest to look at interactions by mating an “a” cell containing mutation Y with an “alpha” cell containing mutation Z (provided none of these Y and Z mutations results in dead or very sick cells). When looking at two (or more!) different mutations together the results are more diverse and can provide more information of the relation between two different proteins and their roles in a particular cellular process. It was this yeast species (which is also a widely used "brewing yeast" to make cider and beer!) that was first used to elucidate the role of specific histone modifications, as one could make individual mutations and also combo mutants to study interactions between different modifications in the same or different histones.